Glutaraldehyde was increasingly used to improve the stability of the collagen-based biomaterials as cross-linking. To investigate in vitro cytotoxicity of Hydroxyapatite/Collagen (HA/COL) composite with glutaraldehyde (GA) as cross-link reagent, which was abbreviated as cHA/COL, the mouse fibroblasts L929 cell was selected. The method of MTT colorimetric assay was adopted to evaluate the availability of cells. After cultured for 24h、 48h, the attachment and morphology of L929 cell on the surface of this material were observed by scanning electron microscopy (SEM) and fluorescent microscopy. MTT analysis indicated L929 cells cultured on cHA/COL had a perfect growth capacity. Cells proliferated well and cell colonization was observed on the materials by scanning electron microscopy and fluorescent microscopy. It demonstrated that cHA/COL composite exhibited good cytocompatibility and might be suitable as bone substitutes. The result provided the evidence for further animal experiment in vivo.